Reprogramming of differentiated somatic cells to have a more undifferentiated stem cell-like state allow scientists to produce induced pluripotent stem cells (iPSCs). IPSCs derived from differentiated somatic cells of patients are potentially a powerful tool for biomedical research and may provide a source of cells for replacement therapies. However, the efficiency of producing and cloning such cells is low and the process can take more than four weeks. Human cell-based IPSC technology also does not present the ethical concerns of human embryonic stem cell technology, which is the only other technology that produces pluripotent stem cells.
Current methods of reprogramming of differentiated somatic cells involve increasing the amount of specific master transcription factors such as KLF4, OCT3/4, c-MYC, NANOG, LIN28, and SOX2 in the somatic cell. These master transcription factors are typically found in undifferentiated cells. They control the expression of other genes, many of which are themselves transcription factors that function to maintain a cell in the undifferentiated state. Currently, there are two main methods of increasing the amount of these master transcription factors: (1) by introducing exogenous genes of the master transcription factors using retroviruses, lentiviruses, or transfection of non-viral plasmids into the somatic cell thereby increasing the cellular expression; and (2) by introducing exogenous proteins of the master transcription factors into the somatic cell through certain transport channels of the cell. A minimum of three master transcription factors is required for reprogramming a differentiated somatic cell to a undifferentiated stem cell-like state, for example, by introducing OCT3/4 and SOX2 with either KLF4 and c-MYC or NANOG and LIN28. Therefore, to reprogram a somatic cell, at least several different exogenous genes or proteins need to be introduced into the cell.
One factor in determining the efficiency of obtaining a reprogrammed somatic cell is the success of introducing the exogenous genes or proteins into the somatic cell and the time that is required to isolate such a reprogrammed cell. Transformation efficiency of a single plasmid into a cell can be as low as less than one transformant cell for every 10,000 cells. The transformation efficiency for at least three independent plasmids representing three master transcription factors can be as low as one in 1012 cells. The use of virus-mediated transfection has improved the transduction efficiency somewhat. The average success rate of producing iPSCs by the virus-mediate method is roughly one in 10,000 cells and takes about four weeks from start to finish.
In addition, there are several problems associated with the current methods: for example, introduction of viral genes and gene products, integration of the exogenous genes or viral genes into the somatic cell genome and non-uniform transfection and/or expression of the exogenous genes and proteins. Therefore, methods that improve the rate and efficiency of producing reprogrammed somatic cells, preferably without transfection of exogenous genes or proteins, are desired, as they are crucial for enabling evaluations of the biomedical value of iPSCs.